Xylitol, which is a naturally occurring sugar alcohol, is a promising low-calorie sweetener because it has lower calories but exhibits comparable sweetness compared with sucrose. In addition, because of its anti-dental caries property, it can be a dental caries preventive sweetener. Furthermore, because xylitol does not elevate glucose level, it has been utilized for fluid therapy in the treatment of diabetes mellitus. For these reasons, it is expected that the demand of xylitol will increase in future.
The current industrial production of xylitol mainly relies on hydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,285. D-Xylose used as a raw material is obtained by hydrolysis of plant materials such as trees, straws, corn cobs, oat hulls and other xylan-rich materials.
However, such D-xylose produced from hydrolysis of plant materials suffers from a drawback that it is rather expensive, and it is arisen from high production cost. For example, the low yield of the hydrolysis treatment of plant materials leads to low purity of the produced D-xylitol. Therefore, the acid used for the hydrolysis and the dyes must be removed by ion exchange treatment after the hydrolysis treatment, and the resulting D-xylose must be further crystallized to remove other hemicellulose saccharides. In order to obtain D-xylose suitable for foodstuffs, further purification would be required. Such ion exchange treatment and crystallization treatment invite the increase of production cost.
Therefore, several methods for producing xylitol have been developed, which utilize readily available raw materials and generate little waste. For example, there have been developed methods for producing xylitol utilizing other pentitols as a starting material. One of such readily available pentitols is D-arabitol, and D-arabitol can be produced by using-yeast (Can. J. Microbiol., 31, 1985, 467-471; and J. Gen. Microbiol., 139, 1993, 1047-54).
Thus, several methods for producing xylitol that utilize D-arabitol as a raw material have been developed. One method has been reported in Applied Microbiology, 18, 1969, 1031-1035, wherein D-arabitol is produced from glucose by fermentation using Debaryomyces hansenli ATCC20121, then converted into D-xylulose using Acetobacter suboxydans, and the D-xylulose is converted into xylitol by the action of Candida guilliermondii var. soya.
EP 403 392A and EP421 882A disclose methods which comprise producing D-arabitol by fermentation using an osmosis-resistant yeast, then converting D-arabitol into D-xylulose using a bacterium belonging to the genus Acetobacter, Gluconobacter, or Klebsiella, forming a mixture of xylose and D-xylulose from the D-xylulose by the action of glucose (xylose) isomerase, and converting the produced mixture of xylose and D-xylulose into xylitol by hydrogenation. There is also disclosed the production of xylitol comprising preliminarily concentrating xylose in the mixture of xylose and D-xylulose and converting the concentrated xylose into xylitol by hydrogenation.
While those methods for the production of xylitol utilizing D-arabitol as a starting material mentioned above can produce xylitol with a relatively high yield, however, they suffer from a drawback that they requires multiple reaction steps, and hence the processes should become complicated. Therefore, they have not been economically acceptable.
On the other hand, breeding of xylitol fermenting microorganisms has been attempted by using genetic manipulation techniques. International Publication WO94/10325 discloses production of xylitol from glucose through fermentation by using a recombinant microorganism obtained by introducing an arabitol dehydrogenase gene derived from a bacterium belonging to the genus Klebsiella and a xylitol dehydrogenase gene derived from a bacterium belonging to the genus Pichia into an arabitol fermenting microorganism (yeast belonging to the genus Candida, Torulopsis, or Zygosaccharomyces).
However, such breeding of xylitol fermenting microorganisms by using genetic manipulation techniques as mentioned above is not considered to be completed as a practical means.
By the way, xylitol dehydrogenase is an enzyme that catalyzes the reaction producing xylitol from xylulose, and its presence has been known in various organisms. For example, there has been known the presence of xylitol dehydrogenase in yeast species such as Pichia stipitis (J. Ferment. Bioeng., 67, 25 (1989)), Pachysolen tannophilus (J. Ferment. Technol., 64, 219 (1986)), Candida shehatae (Appl. Biochem. Biotech., 26, 197 (1990)), Candida parapsilosis (Biotechnol. Bioeng., 58, 440 (1998)), Debaryomyces hansenli (Appl. Biochem. Biotech., 56, 79 (1996)), and Pullularia pullulans (An. Acad. Brasil. Cienc., 53, 183 (1981)), filamentous bacteria such as Aspergillus niger (Microbiology, 140, 1679 (1994)) and Neurospora crassa (FEMS Microbiol. Lett., 146, 79 (1997)), algae such as Galdieria sulphuraria (Planta, 202, 487 (1997)), bacteria such as Morgannela morganil (J. Bacteriol., 162, 845 (1985)), and the like.
As for the xylitol dehydrogenase gene, there have been reported nucleotide sequences of the gene derived from Pichia stipitis (FEBS Lett., 324, 9 (1993)) and Morgannela morganii (DDBJ/GenBank/EMBL accession No. L34345).
However, xylitol dehydrogenase derived from acetic acid bacteria and its gene have not been known so far even for their presence itself.